Transglutaminases (TGases) catalyze the formation of a cross-link between a donor amide group of a protein-bound glutamine residue and an acceptor epsilon-NH2 of a protein-bound lysine residue. This cross-link is an isopeptide bond that cannot be cleaved in vertebrate organisms. The net result therefore is the formation of a permanent, stable, insoluble macromolecular protein complex. In the epidermis and other stratified squamous epithelia, several of the nine known TGase enzymes are expressed. In particular, TGases 1, 2 and 3 cross-link a variety of defined structural proteins to form the cornified cell envelope which is a principal component of epithelial barrier function. We are studying in detail each of these enzymes, and their roles in diseases. Transglutaminase 1 The TGase 1 enzyme in cultured keratinocytes or foreskin epidermal cells is complex since it exists in multiple soluble and membrane-bound full-length as well as proteolytically-processed forms. Most of the enzyme is membrane bound by way of myristate and anchorages on the amino-terminal segment which is unique to the TGase 1 enzyme. The various forms display wide variations in specific activities, but these are difficult to measure because the enzyme is inherently unstable and easily degraded by proteolysis. To address structural and functional questions, we have been successful in expression in baculovirus systems. Previous work from this laboratory has shown that mutations in the TGM1 gene, encoding the TGase 1 enzyme, cause the autosomal recessive disorder lamellar ichthyosis. We examined its molecular basis in a Japanese family and reported two novel TGM1 mutations (R348X, and Y365D). Molecular docking studies revealed that the LI phenotype of this proband can be explained by the prediction that each of the maternally and paternally derived mutations will result in loss of enzyme activity. This study was published. In another investigation of this kind, two self-healing collodion baby siblings with markedly diminished epidermal TGM1 activity we were found to have compound heterozygous TGM1 mutations G278R and D490G. Molecular modeling and biochemical assays of mutant proteins under elevated hydrostatic pressure suggest significantly reduced activity in G278R and a chelation of water molecules in D490G that locks the mutated enzyme in an inactive trans conformation in utero. After birth these water molecules are removed and the enzyme is predicted to isomerize back to a partially active cis form, explaining the dramatic improvement of this skin condition. This study was also published We have continued to perform structural analyses of TGase 3 by X-ray diffraction. In this way, we hope to gain a better understanding of the role of this enzyme in the skin. TGase 3 is expressed in many epithelial cell types, initially as an inactive pro-enzyme, that requires proteolytic activation by specific cleavage. To this end, we developed methods for the large-scale expression and purification of several forms of TGase 3 in the baculovirus system. These include the pro-enzyme, activated enzyme, and the 50 kDa active form. Over the past year, we focussed on regulatory effect of divalent cation binding. Specifically, we solved the structures of three forms of TGM3 in the presence of Ca2+ and/or Mg2+ which provide new insights on the contribution of each Ca2+ ion to activation and activity. First, we found that Ca2+ ion in site one can be exchanged with difficulty and it has a binding affinity of Kd= 0.3 mM, which suggests it is important for the stabilization of the enzyme. Site two can be occupied by some lanthanides but only Ca2+ of the Group 2 family of alkali earth metals, and its occupancy is required for activity. Site three can be occupied by some lanthanides, Ca2+ or Mg2+; however, when Mg2+ is present, the enzyme is inactive, and the channel is closed. Thus Ca2+ binding in both sites two and three cooperate in opening the channel. We speculate that manipulation of the channel opening could be controlled by intracellular cation levels. Together, these data have important implications for reaction mechanism of the enzyme: the opening of a channel perhaps controls access to and manipulation of substrates at the active site.